Screening, DNA libraries

The collection of colonies produced is referred to as a genomic DNA library. The library must be screened with a radioactive probe to identify the colony with the desired restriction fragment (see Screening DNA Libraries). 

In the electronics indnstry, a Pentinm compnter chip has hundreds of millions of transistors and only a hundred pins in and out. If each transistor had to be addressed individually, it would be impossible to have such a chip. Microfluidic systems are similarly easy to control n fluid lines can be controlled by 21og n control hues. Additionally, the pressure to actuate a valve depends on the width of the control line, so by choosing our pressure carefully, one thinner fluid line can be closed while the wider fluid lines remain open due to insufficient pressure. This idea has been used to develop microfluidic systems that can screen enzymatic libraries and perform in vitro transcription translation of DNA to protein in approximately 30 minutes. 

This method is often used in screening gene libraries made from sheared genomic DNA or from cDNA. There is, however, the technical difficulty of deciding on the sequence to be used in the probe, if the cloned gene s sequence is not yet known. Fortunately, there are some ways around this problem. 

A potentially general method of identifying a probe is, first, to purify a protein of interest by chromatography (qv) or electrophoresis. Then a partial amino acid sequence of the protein is determined chemically (see Amino acids). The amino acid sequence is used to predict likely short DNA sequences which direct the synthesis of the protein sequence. Because the genetic code uses redundant codons to direct the synthesis of some amino acids, the predicted probe is unlikely to be unique. The least redundant sequence of 25—30 nucleotides is synthesized chemically as a mixture. The mixed probe is used to screen the library and the identified clones further screened, either with another probe reverse-translated from the known amino acid sequence or by directly sequencing the clones. Whereas not all recombinant clones encode the protein of interest, reiterative screening allows identification of the correct DNA recombinant. 

A single-stranded DNA library was also screened against the HIV-1 RT. IQ values for the selected DNA sequences were in the nanomolar range and they inhibited the DNA polymerase activity of this enzyme with a K, as low as 1 nM (Schneider et al., 1995). The best DNA aptamer folds as a hairpin with an internal loop and competes with the RNA pseudo-knot for RT binding. These two ligands share very little structural similarity. 

Complementary DNAs (cDNA) are DNA copies of mRNAs. Reverse transcriptase is the RNA-directed DNA polymerase that synthesizes DNA strand, using purified mRNA as the template. DNA polymerase is then used to copy the DNA strand forming a double-stranded cDNA, which is cloned into a suitable vector. Once a cDNA derived from a particular gene has been identified, the cDNA becomes an effective probe for screening genomic libraries (Cowell and Austin, 1997). Annotated human cDNA sequences can be accessed from HUNT at http // www.hri.co.jp/HUNT (Yudate, 2001). 

---